The mitogen-activated protein kinases (MAPKs) are inactivated by direct dephosphorylation by a family of enzymes known as MAPK phosphatases (MKPs). The MKPs exert unique physiological outcomes by acting as signaling nodes that integrate multiple MAPK activities. The MAPKs are essential positive regulators of regenerative myogenesis. A complete understanding of how the MAPKs are inactivated during regenerative myogenesis has yet to be achieved. Muscle stem cells are required for regenerative myogenesis and their dysfunction causes degenerative skeletal muscle disease. We have identified MKP5 as an essential MKP that opposes MAPK signaling in skeletal muscle and maintains muscle stem cell quiescence. MKP5 deletion in a mouse model of dystrophic muscle disease ameliorates muscular dystrophy. Hence, MKP5 represents a potential therapeutic target for the treatment of dystrophic muscle disease. The degeneration of skeletal muscle function in congenital dystrophic muscle disease leads to death in young male adults and there is neither a cure nor treatment for this disease. We propose a multi-disciplinary plan encompassing the use of mouse genetics, cell biology, phosphoproteomics, structural biology and chemical biology strategies towards defining MKP5 function in muscle stem cells, skeletal muscle function and a potential therapeutic target for the treatment of degenerative skeletal muscle disease. Aim 1 will define the genetic link between MKP5 and JNK dephosphorylation in regenerative myogenesis. How MKP5 participates in maintaining myofiber homeostasis through control of mitochondrial function will be defined. Aim 2 will employ phosphoproteomic strategies to identify and characterize MKP5-regulated MAPK substrates involved in muscle stem cell function and myofiber integrity. Aim 3, will utilize chemical biology approaches to identify MKP5 small molecule inhibitors. Identified MKP5 inhibitors will be used to provide proof-of- principle for the validity of MKP5 inhibition as a therapeutic target for dystrophic muscle disease. If successful, this work will lay the foundation for new avenues of investigation towards MKP5 as a target for the treatment of degenerative skeletal muscle disease.